The present invention relates to the field of GABA receptor structure and function. In one aspect, the present invention relates to impairment of the gamma-aminobutyric acid (GABA) receptors in parasitic nematodes, for the purpose of crop protection and/or soil treatment. The invention includes mutated nematode GABA receptor subunits, nematode GABA neuromuscular junction receptor complexes, nucleic acids which encode the mutated and functional receptor complexes, antibodies which selectively bind the GABA receptor complexes and/or subunits, and assays for compounds which adversely affect nematode GABA neuromuscular junction receptor function. The present invention therefore relates broadly to recombinant DNA technology, molecular biology tools, and crop protection and/or soil treatment.
The present invention also relates to the fields of GABA neurotransmitter research, particularly with regard to drug discovery. In that regard, the invention includes assays for substances which affect GABA receptors, as well as the tools necessary to conduct such assays, such as nucleic acids, amino acids, antibodies, vectors and cell lines.
Nematodes (nemaxe2x80x94thread; oidesxe2x80x94resembling), which are unsegmented roundworms with elongated, fusiform, or saclike bodies covered with cuticle, are virtually ubiquitous in nature, inhabiting soil, water and plants, and are importantly involved in a wide range of animal and plant parasitic diseases.
The roundworm parasites of mammals belong to the phylum Nemathelminthes. The roundworms include the hookworm (e.g. Necator americanus and Ancylostoma duodenale), roundworm (e.g. the common roundworm Ascaris lumbricoides), whipworm (e.g. Trichuris trichiura), and the pinworm or threadworm (e.g. Enterobius vermicularus), as well as Strongyloides stercoralis, Trichinella spiralis and the filarial worm Wuchereria bancrofti. Other important roundworm parasites include Ancylostoma caninum (infections of man), Strongylus vulgaris (infections of horses), Trichostrongylus colubriformis, Ostertagia circumcincta (infections of sheep and goats), Haemonchus contortus (infections of sheep and goats), Ostertagia ostertagi, Haemonchus placei (infections of cattle), Ascaris suum (infections of pigs), Toxascaris leonina or Uncinaria stenocephala (infections of dogs), Toxocara spp (circulatory infections of man) and Dirofilaria immitis (circulatory infections of cats and dogs).
Even when symptom-free, parasitic worm infections are harmful to the host animal for a number of reasons; e.g. they deprive the host of food, injure organs or obstruct ducts, may elaborate substances toxic to the host, and provide a port of entry for other organisms. In other cases, the host may be a species raised for food and the parasite may be transmitted upon eating to infect the ingesting animal. It is highly desirable to eliminate such parasites as soon as they have been discovered.
More commonly, such infections are not symptom-free. Helminth infections of mammals, particularly by parasitic nematodes, are a source of great economic loss, especially of livestock and pets, e.g. sheep, cattle, horses, pigs, goats, dogs, cats, and birds, especially poultry (see CSIRO/BAE Reportxe2x80x94xe2x80x9cSocio-economic Developments and Trends in the Agricultural Sector: Implications for Future Researchxe2x80x9d). These animals must be regularly treated with anthelminthic chemicals in order to keep such infections under control, or else the disease may result in anaemia, diarrhoea, dehydration, loss of appetite, and even death.
The only currently available means for controlling helminth infections is with the use of anthelminthic chemicals, but these are only effective against resident worms present at the time of treatment. Therefore, treatment must be continuous since the animals are constantly exposed to infection; e.g. anthelminthic treatment with diethylcarbamazine is required every day or every other day most of the year to control Dirofilaria immitis or the dog heartworm. This is an expensive and labour intensive procedure. Due to the widespread use of anthelminthic chemicals, the worms may develop resistance and so new and more potent classes of chemicals must be developed. An alternative approach is clearly desirable.
The accepted methodology for pharmaceutical control of nematodes has centered around the use of the drug benzimidazole and its congeners. The use of these drugs on a wide scale has led to many instances of resistance among nematode populations (Prichard et al., 1980; Coles, 1986). There are more than 100,000 described species of nematodes.
Other options for a drug treatment include employing an avermectin, pyrantel, morantel, closantel, praziquantel etc. Benzimidazole(s) or benzimidazole prodrug treatments include oxfendazole, thiabendazole, albendazole, cambenazole, fenbendazole, flubendazole, mebendazole, oxibendazole, parbendazole, thiophanate, febantel and netobimin.
Nematodes thrive in virtually all environments throughout the world and are one of the largest and most diverse groups of multicellular organisms. Many species are parasites of agronomic crops, but other species are beneficial to agriculture. Nematodes that parasitize plants cause an estimated $8 billion annual loss (12%) to U.S. growers and nearly $78 billion loss globally. For example, the soybean cyst nematode causes annual losses in the North Central Region of the U.S. amounting to $267 million.
Traditional nematode reduction methods usually rely on a combination of petroleum byproduct soil treatments and crop rotation. Stricter environmental regulations are forcasted to limit the use of petroleum by-products, and threaten to impact agriculture if no safer alternatives are found or invented. A review of the impact of nematodes on crops can be found in: Hussey, R. Plant and Soil Nematodes: Societal Impact and Focus for the Future and can be obtained by writing to Department of Plant Pathology, University of Georgia, Athens, Ga. USA 30602-7274.
The gamma-aminobutyric acid receptors (GABA receptors) are the most abundant inhibitory receptor in the mammalian brain. They are comprised of a heteropolymeric structure that form a chloride ion channel, and contain multiple recognition sites for the binding of molecules. The binding of GABA to its specific recognition site on the a GABA receptor opens the ion channel and allows chloride ions to flow into the nerve cell. This action hyperpolarizes the cell membrane of that neuron and thereby makes the cell less reactive to excitatory stimuli. The chloride ion current may also be regulated by various drugs that serve as positive or negative modulators of the GABA receptor (Puia, G. et al. Molecular Pharm. 1991, 39, 691).
Many clinical conditions are thought to arise, in part, from the imbalance between neurotransmission of GABA and those of other neurotransmitters. These conditions include Huntington""s chorea, Parkinson""s disease, spasticity, epilepsy, schizophrenia and tardive dyskinesia. Decreased GABA activity appears to contribute to the pathogenesis of these diseases. In addition, analgesia and satiety are thought to be regulated by GABA activity. Methods of modifying GABAergic neurotransmission are therefore desirable in order to modify these conditions.
The so-called benzodiazepine (BZD) receptor is a site for such allosteric modulators on one class of the GABA receptor, the GABA-A receptor. This site mediates two opposing effects, one that amplifies the action of GABA (xe2x80x9cpositivexe2x80x9d efficacy) and the other that reduces the action of GABA (xe2x80x9cnegativexe2x80x9d efficacy). Agents facilitating GABA-receptor/chloride ion-channel functions via the BZD site are referred to as agonists, while agents reducing such function are referred to as inverse agonists. Antagonists at this site block the effects of agonists or inverse agonists by competitively inhibiting their binding. It is thus possible to have a series of compounds in which members equally bind to the BZD site but have equal and opposite regulatory effects on the GABA-A receptor/chloride ion channel. Also, within the series a continuum of activity is possible (Takada, S. et al. J. Med. Chem. 1988, 31, 1738). Thus, BZD receptor ligands can induce a wide spectrum of pharmacological effects ranging from muscle relaxant, hypnotic, sedative, anxiolytic, and anticonvulsant activities, produced by full or partial agonists (xe2x80x9cpositivexe2x80x9d), to the proconvulsant, anti-inebriant, and anxiogenic activities, produced by inverse agonists (xe2x80x9cnegativexe2x80x9d). (A further understanding of this area can be gleaned from: Mohler, H. Arzneim.-Forsch./Drug Res.1992, 42 (2a), 211; Haefely, W. et al., Advances in Drug Research, Academic Press, vol. 14, 1985, pp. 165-322; Skolnick, P. et al., GABA and Benzodiazepine Receptors, Squires, R., Ed., 1987, pp. 99-102 and references cited therein.)
The fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV.TM.), published in 1994 by the American Psychiatric Association, Washington, D.C., defines anxiety and related disorders. These are panic disorder without agoraphobia, panic disorder with agoraphobia, agoraphobia without history of panic disorder, specific phobia, social phobia, obsessive-compulsive disorder, post-traumatic stress disorder, acute stress disorder, generalized anxiety disorder, anxiety disorder due to a general medical condition, substance-induced anxiety disorder and anxiety disorder not otherwise specified.
Anxiety disorders are generally treated by counseling or with drugs. Classes of drugs which are widely prescribed for the treatment of anxiety disorders include the benzodiazepines (such as diazepam) and buspirone hydrochloride.
Several animal models have been developed which are recognized in the art as being predictive of anxiolytic activity. These include the fear-potentiated startle model, described by Davis in Psychopharmacology 62:1; 1979, Behav. Neurosci. 100:814;1986 and TiPS, January 1992 Vol. 13, 35-41, the elevated plus model described by Lister in Psychopharmacol. 92:180-185; 1987, and the well-known punishedxe2x80x94responding (conflict) model, described, for example, in xe2x80x9cPsychopharmacology of Anxiolytics and Antidepressantsxe2x80x9d, edited by S. E. File, pp. 131-153, Raven Press, New York, 1991.
Citation of the above documents is not intended as an admission that any of the foregoing is prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of the documents.
The present invention therefore provides nematode neuromuscular juntion GABA receptor complexes of the formula I
A-Bxe2x80x83xe2x80x83Formula I
wherein A an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence which is encoded by a nucleic acid sequence which has at least 80% identity to SEQ ID NO 2; wherein said identity can be determined using the DNAsis computer program and default parameters; and
(b) an amino acid sequence which has at least 80% identity to SEQ ID NO 1; wherein said identity can be determined using the DNAsis computer program and default parameters; and
wherein B is an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence which is encoded by a nucleic acid sequence which has at least 80% identity to SEQ ID NO 4; wherein said identity can be determined using the DNAsis computer program and default parameters; and
(b) an amino acid sequence which has at least 80% identity to SEQ ID NO 3; wherein said identity can be determined using the DNAsis computer program and default parameters; and
wherein A-B is a heteropentamer which comprises 1 to 4 A amino acid sequences and 1 to 4 B amino acid sequences.
Also provided are homopentamer nematode neuromuscular juntion GABA receptor complexes of the formula II
B5xe2x80x83xe2x80x83Formula II
wherein B is an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence which is encoded by a nucleic acid sequence which has at least 80% identity to SEQ ID NO 4; wherein said identity can be determined using the DNAsis computer program and default parameters; and
(b) an amino acid sequence which has at least 80% identity to SEQ ID NO 3; wherein said identity can be determined using the DNAsis computer program and default parameters.
Also provided are nematode neuromuscular junction GABA receptor complex subunits, comprising an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence which is encoded by a nucleic acid sequence which has at least 80% identity to SEQ ID NO 2; wherein said identity can be determined using the DNAsis computer program and default parameters; and
(b) an amino acid sequence which has at least 80% identity to SEQ ID NO 1; wherein said identity can be determined using the DNAsis computer program and default parameters.
Also provided are nematode neuromuscular junction GABA receptor complex subunits, comprising an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence which is encoded by a nucleic acid sequence which has at least 80% identity to SEQ ID NO 4; wherein said identity can be determined using the DNAsis computer program and default parameters; and
(b) an amino acid sequence which has at least 80% identity to SEQ ID NO 3; wherein said identity can be determined using the DNAsis computer program and default parameters.
Also provided are nucleic acid compound which encodes a nematode neuromuscular junction GABA receptor complex subunits, comprising:
(a) a nucleic acid sequence which has at least 80% identity to SEQ ID NO 2; wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid which encodes an amino acid sequence which has at least 80% identity to SEQ ID NO 1; wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which is an allelic variant of SEQ ID NO 2; and
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); and a nucleic acid sequence of (c).
Also provided are nucleic acid compounds which encode a nematode neuromuscular junction GABA receptor complex subunit, comprising a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which has at least 80% identity to SEQ ID NO 4; wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid which encodes an amino acid sequence which has at least 80% identity to SEQ ID NO 3; wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which is an allelic variant of SEQ ID NO 4; and
(d) a nucleic acid sequence fully complementary to a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence of (a); a nucleic acid sequence of (b); and a nucleic acid sequence of (c).
Also provided are nucleic acid compounds which encode a nematode neuromuscular junction GABA receptor complex, comprising a first and second nucleic acid sequence, wherein said first nucleic acid sequence is selected from the group consisting of:
(a) a nucleic acid sequence which has at least 80% identity to SEQ ID NO 2; wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid which encodes an amino acid sequence which has at least 80% identity to SEQ ID NO 1; wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which is an allelic variant of SEQ ID NO 2; and
(d) a nucleic acid sequence which has at least 80% identity to SEQ ID NO 4; wherein said identity can be determined using the DNAsis computer program and default parameters;
(e) a nucleic acid which encodes an amino acid sequence which has at least 80% identity to SEQ ID NO 3; wherein said identity can be determined using the DNAsis computer program and default parameters;
(f) a nucleic acid sequence which is an allelic variant of SEQ ID NO 4; and
wherein said second nucleic acid sequence is selected from the group consisting of:
(a) a nucleic acid sequence which has at least 80% identity to SEQ ID NO 4; wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid which encodes an amino acid sequence which has at least 80% identity to SEQ ID NO 3; wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which is an allelic variant of SEQ ID NO 4.
Moreover, there are provided isolated antibodies selective for a GABA receptor complexes of the present invention.
In addition, there are provided methods to determine a test substance""s ability to interact with a nematode neuromuscular junction GABA receptor complex, comprising contacting a receptor complex of the present invention with a test substance and determining whether said test substance and said receptor complex interact.
Other methods include those to detect GABA receptors in a test sample, comprising: (a) immobilizing a test sample on a substrate; (b) contacting the test sample with an antibody of the present invention under conditions suitable for formation of a GABA receptor:antibody complex bound to the substrate; (c) removing non-bound material from the substrate under conditions that retain GABA:antibody complex binding to the substrate; and (d) detecting the presence of the GABA receptor:antibody complex.
Preferred isolated nucleic acid compounds of the present invention are those which comprise a nucleic acid sequence selected from the group consisting of: SEQ ID NO 2; SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQ ID NO 10; and SEQ ID NO 12.
Preferred isolated amino acid compound of the present invention are those which comprise a nucleic acid sequence selected from the group consisting of: SEQ ID NO 1; SEQ ID NO 3, SEQ NO 5; SEQ ID NO 7; SEQ ID NO 9; SEQ ID NO 11.
Moreover, for the purposes of the present invention, the term xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d entity refers to one or more of that entity; for example, xe2x80x9ca proteinxe2x80x9d or xe2x80x9ca nucleic acid moleculexe2x80x9d refers to one or more of those compounds or at least one compound. As such, the terms xe2x80x9caxe2x80x9d (or xe2x80x9canxe2x80x9d), xe2x80x9cone or morexe2x80x9d and xe2x80x9cat least onexe2x80x9d can be used interchangeably herein. It is also to be noted that the terms xe2x80x9ccomprisingxe2x80x9d, xe2x80x9cincludingxe2x80x9d, and xe2x80x9chavingxe2x80x9d can be used interchangeably. Furthermore, a compound xe2x80x9cselected from the group consisting ofxe2x80x9d refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure, protein or nucleic acid molecule is a compound that has been removed from its natural milieu. As such, xe2x80x9cisolatedxe2x80x9d and xe2x80x9cbiologically purexe2x80x9d do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis.